Francisella tularensis (Ft) is a Gram negative, intracellular (macrophage-tropic) bacterium, and the etiologic agent of tularemia. Ft is classified as a Category A agent because it may can be contracted with low inocula by the respiratory route and causes rapid morbidity and mortality if untreated both mice and humans. Ft Live Vaccine Strain (LVS), a type B strain derived from Ft subsp. holarctica, is attenuated for man, but causes a tularemia-like disease in mice when administered intranasally or intraperitoneally. Although Ft LVS was shown to reduce the incidence of laboratory-acquired respiratory tularemia, it is not licensed in the USA because the molecular basis for its attenuation has not been fully elucidated, the strain exhibits phenotypic inconsistencies, and Ft LVS immunization fails to provide complete protection against some subspecies of Ft. Therefore, additional prophylactic products and/or attenuated vaccine strains must be developed for use against possible bioterrorism threats. Our recently published results show that insertional inactivation of the lpxF gene in F. novicida (Fn) resulted in an attenuated strain with an altered lipopolysaccharide (LPS) structure that protected immunized mice against >25,000 LD50 challenge with wild-type (WT) Fn. We now show that a similar mutation in Ft LVS resulted in the same structural modification to the lipid A and was avirulent at >106 LD50 after subcutaneous infection of C57BL/6 and BALB/c mice. The goal of this revised R21 proposal is to develop live, rationally attenuated Ft vaccines based on unmarked deletion of lpxF in both Ft LVS (Type B) and Ft Schu S4 (Type A) strains. After engineering an unmarked lpxF deletion in these two strains, we will confirm the structure of the lipid A from each mutant by mass spectroscopy, and confirm that each mutant is, indeed, attenuated in mice. Additionally, we will test their capacity to protect WT, IFN-r-/-, and TLR2-/- mice against homologous and heterologous challenge. Finally, we will test for the capacity of each mutant strain to induce macrophage gene expression associated with classically activated (microbicidal) vs. alternatively activated (shown previously to facilitate intracytosolic replication) macrophages and for the ability to replicate intracellularly in vitro. At the completion of this study, we expect to have developed a rationally attenuated vaccine candidate that will protect against multiple Ft Type A and Type B Francisella strains.